In vivo and ex vivo mechanisms related to eNOS uncoupling during reperfusion

描述(申请人提供)：再灌注损伤是由内皮功能障碍引起的，其特征是氧化应激增加和内皮衍生的一氧化氮(NO)生物可利用性有限，导致多形核白细胞(PMN)血管黏附和渗透增加，继而导致血管和器官功能障碍。因此，减轻内皮功能障碍可能是限制再灌注损伤的有效途径。内皮型一氧化氮合酶(ENOS)在正常的内皮功能中起着核心作用。然而，当eNOS的重要辅因子四氢生物蝶呤(BH4)在体外被氧化为二氢生物蝶呤(BH2)时，eNOS可以将其产物从产生NO转变为超氧化物(SO)(eNOS解偶联)。到目前为止，控制eNOS解偶联的机制还没有通过实时直接测量体内再灌流过程中的氧化应激来证实。最近，我们建立了一种在大鼠股动脉/静脉缺血/再灌注(I/R)模型中使用特定的H_2O_2或NO微型传感器直接测量股静脉释放NO和H_2O_2(SO的间接指标)的方法。我们的初步结果显示，在同一动物的I/R股静脉中，与非缺血(假)股静脉相比，H_2O_2显著增加。此外，BH4或eNOS抑制剂(如蛋白激酶C epsilon多肽抑制剂(PKC？-))减少，而BH2或eNOS增强子(如PKC？-)减少。激活剂(PKC+)可增加再灌流过程中过氧化氢的释放。此外，BH4还显著增加了股I/R静脉NO的释放。此外，BH4或PKC+增加，而BH2或PKC-减少NO的释放。在我们的体外PMN诱导的心肌I/R损伤模型中，BH4或PKC？-，在再灌流期间单独给予BH2或PKC？-都不能起到心脏保护作用。根据我们的初步结果，我们推测BH4氧化为BH2是再灌流过程中eNOS解偶联的主要原因，也是PKC？+/PKC？-对抗H_2O_2/NO释放和心脏保护作用的主要原因。为了验证这一假设，我们将在BH4、BH2、PKC？+或PKC？-单独或不同组合的情况下执行以下特定目标：1)和2)在我们的股骨I/R模型中，体内测量股I/R静脉和假静脉的过氧化氢或NO释放；3a)、3b)和4)体外测量非缺血大鼠主动脉段的NO释放，体外评估灌流后心脏收缩功能、心脏损伤和PMN积聚。我们预测，BH4+PKC+(即eNOS激活)，而不是BH2+PKC+，将显著降低氧化应激，保护心脏免受再灌注损伤。研究再灌注损伤中eNOS解偶联的体内和体外机制(S)的公共卫生相关性，将对寻找更多的选择性治疗方法，如BH4或BH4联合PKC+，用于器官移植和再灌流期间的心脏病发作患者具有重要影响。与器官移植或心脏病发作后久坐不动的生活方式相比，临床效果将显著更好地恢复器官功能，从而产生更好的生活质量(即有氧运动)。

DESCRIPTION (provided by applicant): Reperfusion injury is initiated by endothelial dysfunction, which is characterized by increased oxidative stress and limited endothelial-derived nitric oxide (NO) bioavailability, leading to enhanced polymorphonuclear leukocyte (PMN) vascular adherence and infiltration following by blood vessel and organ dysfunction. Therefore, attenuation of endothelial dysfunction may be an effective way to limit reperfusion injury. Endothelial NO synthase (eNOS) plays a central role in normal endothelial function. However, eNOS can shift its product profile from producing NO to superoxide (SO) (eNOS uncoupling) when its essential cofactor, tetrahydrobiopterin (BH4) is oxidized to dihydrobiopterin (BH2) in vitro. Until now, the mechanisms that govern eNOS uncoupling have not been demonstrated by real-time direct measuring oxidative stress during reperfusion in vivo. Recently, we developed a direct measurement of NO and H2O2 (indirect index of SO) release from femoral veins in vivo in a rat femoral artery/vein ischemia/reperfusion (I/R) model using specific H2O2 or NO microsensors. Our preliminary results show that H2O2 is significantly increased in I/R femoral veins compared to non-ischemic (sham) femoral veins in the same animal. Moreover, BH4 or an eNOS inhibitor (e.g., protein kinase C epsilon peptide inhibitor (PKC ?-)) decreased, whereas BH2 or an eNOS enhancer (e.g., PKC ? activator (PKC ?+)) increased H2O2 release during reperfusion. Additionally, BH4 also significantly increased NO release from femoral I/R veins. Furthermore, BH4 or PKC?+ increased, whereas BH2 or PKC ?- decreased NO release in non-ischemic rat aortic segments, respectively. In our ex vivo PMN-induced myocardial I/R injury model, BH4 or PKC ?-, neither BH2 nor PKC ?+ provided cardioprotection when given separately during reperfusion. Based on our preliminary results, we hypothesize that the oxidation of BH4 to BH2 is mainly responsible for eNOS uncoupling during reperfusion, and is the primary cause for opposing effects of PKC ?+/PKC ?- on H2O2/NO release and cardioprotection. To test the hypothesis, we will perform the following specific aims in the presence/absence of BH4, BH2, PKC ?+ or PKC ?- alone or in different combinations: 1) and 2) in vivo measurement of H2O2 or NO release from the femoral I/R vein and sham vein in our femoral I/R model; 3a), 3b) and 4) in vitro NO release measurement from isolated non-ischemic rat aortic segments, ex vivo evaluation of postreperfused cardiac contractile function, heart injury and PMN accumulation. We predict that BH4+PKC ?+ (i.e., eNOS activation), not BH2 +PKC ?+ will significantly decrease oxidative stress and protect heart from reperfusion injury. PUBLIC HEALTH RELEVANCE Characterizing the in vivo and ex vivo mechanism(s) responsible for eNOS uncoupling in reperfusion injury would have a significant impact in identifying more selective treatments, such as BH4 or BH4 combined with PKC ?+, for organ transplants and heart attack patients during reperfusion of blood flow. The clinical impact would be significantly better restoration of organ function resulting in a better quality of life (i.e., aerobic exercise) compared to more sedentary life-style after organ transplantation or heart attacks.